Gene 290 (2002) 107–114 www.elsevier.com/locate/gene Rat zinc-fingers and homeoboxes 1 (ZHX1), a nuclear factor-YA-interacting nuclear protein, forms a homodimer Satoko Hiranoa,b, Kazuya Yamadab,c,*, Hiroko Kawatac, Zhangfei Shoub,d, Tetsuya Mizutanib,c, Takashi Yazawab,c, Takashi Kajitanib,c, Toshio Sekiguchib,c, Miki Yoshinob,c, Yousuke Shigematsua, Mitsufumi Mayumia, Kaoru Miyamotob,c aDepartment of Pediatrics, Fukui Medical University, Fukui 910-1193, Japan bDepartment of Biochemistry, Fukui Medical University, Fukui 910-1193, Japan cCREST, JST (Japan Science and Technology), Fukui 910-1193, Japan dDepartment of Urology, Fukui Medical University, Fukui 910-1193, Japan Received 25 December 2001; received in revised form 21 February 2002; accepted 11 March 2002 Received by T. Gojobori Abstract Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Yeast two-hybrid; Transcription factor; Homeobox; Zinc-finger motif; Dimerization; Nuclear localization 1. Introduction these factors on the promoter serves to determine gene- specific transcription. Most transcription factors not only Granulosa cells play important roles in ovarian function bind to the specific cis-acting element but also interact including follicular maturation and ovulation (Richards, with one another. In addition to these DNA-binding 2001). Many granulosa cell genes express proteins in a proteins, DNA-non-binding proteins also regulate the tran- cell type-specific manner, and some of these are also tran- scription of the target genes. These factors, referred to as the scriptionally regulated by gonadotropins, such as follicle co-activators or co-repressors, act by bridging or interfering stimulating hormone and luteinizing hormone. Generally, with interactions among DNA-binding proteins and basic gene transcription is positively or negatively regulated by transcription machinery (Hu and Lazar, 2000; Vo and Good- transcription factors which bind to the regulatory region man, 2001). within the promoter of the gene. Cell type-specific transcrip- Nuclear factor-Y (NF-Y), a ubiquitous transcription tion factors and ubiquitous transcription factors are known factor, binds to an inverted CCAAT nucleotide sequence to exist (Fry and Farnham, 1999). Unique combinations of (Y box, 50-ATTGG-30) and stimulates the transcription of a number of genes (Mantovani, 1999). NF-Y is thought to be Abbreviations: NF-Y, nuclear factor-Y; ZHX1, zinc-fingers and home- essential for cell proliferation stimulated by estradiol in oboxes 1; AD, activation domain; Znf, zinc-finger; HD, homeodomain; kb, MCF-7 cells through the activation of various cell cycle kilobases; DES, diethylstilbestrol; GST, glutathione-S-transferase; DBD, related genes (Wang et al., 1999). NF-Y consists of three DNA-binding domain; GFP, green fluorescence protein; RACE, rapid subunits, YA, YB, and YC, which also interact with other amplification of the cDNA ends; USF, upstream stimulatory factor; NLS, transcription factors (Mantovani, 1999; Yamada et al., nuclear localization signal * Corresponding author. Tel.: 181-776-61-8316; fax: 181-776-61-8102. 2000). We previously cloned human zinc-fingers and home- E-mail address: [email protected] (K. Yamada). oboxes 1 (ZHX1) and serum response factor as proteins 0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S0378-1119(02)00553-X 108 S. Hirano et al. / Gene 290 (2002) 107–114 which interact with the activation domain (AD) of NF-YA reagent was purchased from Roche Molecular Biochemicals (Yamada et al., 1999a,b). Mouse ZHX1 was independently Mannheim (Indianapolis, IN). HEK293 cells, a human reported as an antigen which is recognized by the B92 embryonic kidney cell line, were purchased from the Amer- monoclonal antibody produced by immunization with a ican Type Culture Collection (Manassas, VA). cell lysate of 14F1.1 endothelial-adipose stromal cells (Barthelemy et al., 1996). Both human and mouse ZHX1 2.2. Animals and treatment consist of 873 amino acid residues and contains two Cys2- Kwl:Wistar immature female rats (21 days old) were used His2-type zinc-fingers (Znf) and five homeodomains (HDs) (Barthelemy et al., 1996; Yamada et al., 1999b). It belongs in this study. The rats were treated with 2 mg DES in 0.1 ml to the Znf class of the homeobox protein superfamily (Gehr- sesame oil once daily for 4 days for the preparation of ing et al., 1994). The amino acid sequence between 272 and granulosa cells. For the in vivo study, a 30 IU of eCG 564 that contains the HD1 through HD2 region of the human alone, or 50 IU of hCG after eCG treatment for 48 h were ZHX1 is required for interaction with a glutamine-rich AD administrated and ovaries were removed at the indicated of NF-YA (Yamada et al., 1999a). Northern blot analysis of times. At all times, the animals were treated following poly(A)1-RNA isolated from various human tissues has NIH guidelines. shown that two major ZHX1 transcripts comprised of about 4.5 and 5 kilobases (kb) are expressed ubiquitously 2.3. Plasmids (Yamada et al., 1999b). The human ZHX1 gene is located on The pBSII-hZHX1E/X, pEGFP-C1E1, pGEM-T- chromosome 8q, between markers CHLC.GATA50B06 and hZHX1E, and pGST-ZHX1E/X plasmids has been CHLC.GATA7G07 (Yamada et al., 1999b). described previously (Inazu et al., 1999; Yamada et al., To address the issue of whether ZHX1 interacts with 1999a,b). A 1.9-kb EcoRI/BamHI fragment was isolated protein(s) other than NF-YA in rat granulosa cells, we from the pBSII-hZHX1E/X and subcloned into the EcoRI/ employed a yeast two-hybrid system. Here, we report on BamHI sites of the pGBKT7 and pEGFP-C1E1 plasmids to the molecular cloning and characterization of full-length produce pGBKT7-ZHX1EB and pEGFP-ZHX1EB, respec- rat ZHX1 cDNA. tively. A PCR was carried out using the pGEM-T-hZHX1E as a template and 1ZHX1, 50-CCGGGAATTCATGG- CAAGCAGGCGAAAATC-3 0, and 272ZHX1, 50-GGGAT- 2. Materials and methods TAAGACTTTGGGAATC-30, as primers. PCR conditions have been described previously except for the use of the 2.1. Hormones and materials ExTaq DNA polymerase (Yamada et al., 1999a). The product was subcloned into the pGEM-T Easy vector to Diethylstilbestrol (DES) was purchased from Sigma produce pGEM-T Easy-ZHX1 (1–271). A 0.9-kb EcoRI Chemical Co. (St. Louis, MO) and hCG was obtained fragment from the pGEM-T Easy-ZHX1 (1–271) was from Sankyo Co., Ltd. (Tokyo, Japan). The eCG was subcloned into the EcoRI site of the pGST-ZHX1E/X, obtained from Teikokuzouki, Inc. (Tokyo, Japan). The pGBKT7-ZHX1EB and pEGFP-ZHX1EB to obtain pGST- yeast two-hybrid system, rat brain marathon complementary ZHX1(1–873), pGBKT7-ZHX1 (1–873) and pEGFP-ZHX1 DNA (cDNA), Advantage 2 polymerase chain reaction (1–873), respectively. Each plasmid expresses the entire (PCR) kit, rat Multiple Tissue Northern blot, ExpressHyb coding region of the human ZHX1 fused to glutathione-S- hybridization solution and pEGFP-C1 vector were transferase (GST), to DNA-binding domain (DBD) of the purchased from CLONTECH (Palo Alto, CA). The ExTaq yeast GAL4 transcription factor, or to the green fluores- DNA polymerase and BcaBest DNA labeling kit were cence protein (GFP). The nucleotide sequences of all plas- obtained from Takara BIOMEDICALS (Kyoto, Japan). mids were determined. The pGEM-T Easy vector and T7 TNT Quick-coupled tran- scription/translation system were purchased from Promega 2.4. Yeast two-hybrid system and library screening (Madison, WI). The Big Dye terminator FS cycle sequen- cing kit was purchased from Applied Biosystems Japan AH109 yeast cells were transformed with the pGBKT7- (Tokyo, Japan). The pGEX-5X-1 vector, Glutathione- ZHX1 (1–873) plasmid. A TE/LiAc-based high efficiency Sepharose 4B, and 35S-methionine (37 TBq/mmol) were transformation method was used for the yeast transforma- purchased from Amersham Pharmacia Biotech (Cleveland, tion (Yamada et al., 1998). This yeast strain was used as a OH). The TOPP3 cells were obtained from Stratagene (La bait to screen a cDNA library. The construction of the rat Jolla, CA). a-32P dCTP (111 TBq/mmol) was purchased granulosa cell cDNA library has been described previously from NEN Life Science Products (Wilmington, DE). The (Yamada et al., 2001). Approximately eleven million inde- TRIZOL reagent, Superscript II, and LIPOFECTAMINE pendent clones of the granulosa cell cDNA library were PLUS were purchased from Invitrogen (Groningen, Nether- plated on histidine-, tryptophan-, leucine-, and adenine- lands). The QIAGEN plasmid kit was purchased from free synthetic dextrose (SD-His-Trp-Leu-Ade) plates QIAGEN (Hilden, Germany). The FuGENE 6 transfection supplemented with 4 mM 3-aminotriazole and X-a-gal. S. Hirano et al. / Gene 290 (2002) 107–114 109 One hundred and nine positive clones were obtained from out according to the manufacturer’s protocol. Amplified the primary transformants. DNA fragments were subcloned into the pGEM-T Easy vector and their nucleotide sequences were determined.